1. Field of the Invention
This invention relates to a process for the preparation of immobilized glucose isomerase by a method of immobilizing glucose isomerase in the state as contained in the microbial cell as it is.
2. Brief Description of the Prior Art
Hitherto, the industrial application of glucose isomerase was usually carried out by using the whole microbial cells containing glucose isomerase in themselves as enzyme preparation. In the state of native cells, however, the enzyme would dissolve out quite rapidly into the reaction solution, and so the upper limit of repetition times of usage was at most two or three considering from economic aspect.
Since it is required for saving the cost of enzyme to carry out the isomerisation with a smaller dosage of enzyme under a longer period of time, there are many disadvantages in such batch reaction, for examples, the requirement of a larger scale of reaction apparatus, the heavy coloration of isomerized syrup, and the expensive cost of purification of the colored syrup.
When the glucose isomerase is immobilized, glucose solution can be treated continuously by flowing through a layer of the immobilized enzyme in a reaction tower. In this case, the reaction time can be extremely shortened and accordingly the formation of coloring compounds and other impurities will be sharply reduced, and also the reaction apparatus and treating operation can be simplified. Thus a process using immobilized glucose isomerase is extremely advantageous for industrial isomerization of glucose.
Recently there have been examined various attempts for immobilizing glucose isomerase and conducting isomerization in a continuous process by the use of immobilized glucose isomerase, and proposed many methods thereof.
As for the immobilizing method of enzyme there can be cited a method wherein glucose isomerase is extracted from the microbial cells, then absorbed on a carrier such as ion-exchange resin (Japanese Open Laid Patent Specification No. Sho 50-53582) and activated alumina (Japanese Open Laid Patent Specification No. Sho 49-110887), a method wherein the extracted enzyme is entrapped in a synthetic fibre, a method wherein microbial cells containing glucose isomerase are treated with a cross-linking agent to solidify their cell walls, and also to combine with each other by the formation of cross-linkages among them (Japanese Open Laid Patent Specification No. Sho 49-92278), a method wherein microbial cells containing glucose isomerase are adsorbed on a special type of resin (Japanese Open Laid Patent Specification No. Sho 50-6774), or a method wherein microbial cells containing glucose isomerase are entrapped in a polymer, such as acrylamide gel, or collagen.
Among the above methods, those methods wherein glucose isomerase extracted from microbial cells is adsorbed on various carriers require a process for extracting enzyme from cells, because glucose isomerase is an intracellular enzyme, and usually also a process for purifying the extracted enzyme in order to prepare an immobilized enzyme of higher activity. Thus, a severe loss of enzyme is inevitable in these treatments.
Some methods for immobilizing microbial cells containing glucose isomerase have been known publicly in Japanese Open Laid Patent Specification No. Sho 49-92278, U.S. Pat. No. 3838007 and so on.
In Japanese Patent Publication No. Sho 50-37274, the disclosuure relates to a method for insolbilizing acid protease which is produced by a strain of Rhizopus and active in the acidic circumstance. However, when the pH value in the treatment is different from the adequate pH value in the enzymatic reaction, the activity of the enzyme is extremely reduced and the utilization of the preparation can not be made. Accordingly, it is impossible to apply the above method directly for immobilizing glucose isomerase which is produced by a strain of Actinomycetes and enzymatically active in the range of pH from neutral to alkaline, and even if the pH value in the treatment of immobilization is adjusted to a pH within a range where the activity of glucose isomerase is stable, immobilization of glucose isomerase cannot be attained effectively.